RNA tumor viruses replicate via synthesis and integration of the provirus, a DNA copy of the high molecular weight genomic RNA. I propose to study the mechanism by which synthesis of the DNA copy is initiated in vitro, and the structure of the integrated provirus, using Rous sacroma virus (RSV). We have shown that RSV DNA synthesis is initiated by extension of a primer RNA, tRNA Trp of the host cell. We intend to study the primer-template interaction in an effort to determine how the primer RNA works, how it binds to the template and where on the template DNA synthesis actually starts. To do this, we shall fractionate and chemically modify tRNA Trp and test which parts of the molecule are required for template binding and for priming activity. We shall likewise fractionate the template. By hybridization of specific short cDNA products to fragments of the template RNA we intend to determine where DNA synthesis is initiated. The structure of the integrated provirus and its location relative to endogenous viral sequences will be studied using in situ hybridization of RNA and cDNA probes to restriction enzyme fragments of cell DNA. Experiments are designed to show whether integration occurs at preferred sites in the pre-provirus and the host DNAs, and if so, where that site is located in the pre-provirus. Using a novel method of restriction enzyme fragment mapping we intend to determine restriction fragment maps of the provirus. We also plan to determine the map of the HAE III endonuclease fragments of in vitro synthesized cDNA. The mapping techniques discussed here should be applicable to the study of other eukaryotic gene structures even when the gene themselves cannot be purified.